Group of nucleic acid fragments for prevention of HIV infection or AIDS and the usage thereof

ABSTRACT

The invention provides a group of nucleic acid fragments, shown in the sequence listing, for prevention of HIV infection or AIDS and the usage thereof. In the invention, a series of RNA fragments, which are highly homogenous to all the published HIV gene sequences, were obtained by homology compare. The double-stranded RNA (dsRNA) derived from these fragments can effectively inhibit the expression of the HIV genes. The RNA transcribed by plasmid, also can suppress the expression of the HIV in the cell. After the adenovirus or associated virus which carry DNA corresponding above RNA infect the cell, the transcription dsRNA can inhibit the expression of the HIV genes.

TECHNICAL FIELD

The invention is regarded to a set of oligo-nucleotides against HIVinfection and its application in the prevention and treatment ofAcquired Immune Deficiency Syndrome (AIDS).

TECHNOLOGY BACKGROUND

Recent findings proved that short double strand RNA function asinterference RNA in a variety of mammalian cells, and gene expressioncan be specifically knocked down. Viral gene (including HIV) expressioncan be knocked down by this pathway. Due to the high frequency ofmutation in HIV genome, most of the interfere RNA can knock down thegene expression of specific isolates and can not be used as a universalapproach in gene therapy of AIDS.

INVENTION DISCLOSURE

The purpose of the invention is to provide a set of nucleotides for theprevention of HIV infection and treatment of AIDS.

The other purpose is to provide the application of the oligo-nucleotidesmentioned above.

For the purposes, following approaches were employed.

A set of RNA sequences shown thereafter, or any fragments from thesequences, which demonstrate anti-HIV infection activity and be employedin prevention and treatment of AIDS. The nucleotides include singlestrand RNA, any fragment derived from the sequences, or double strandRNA derived by annealing of the sequences with its complementssequences.

(SEQ ID NO: 1) (1) aucaaugaggaagcugcagaaugg; (SEQ ID NO: 2) (2)gggaagugacauagcaggaacuacuag; (SEQ ID NO: 3) (3)uaaauaaaauaguaagaauguauagcccu; (SEQ ID NO: 4) (4) uaugggguaccugugugga;(SEQ ID NO: 5) (5) gccaauucccauacauuauugugc; (SEQ ID NO: 6) (6)uuaaauggcagucuagcagaa; (SEQ ID NO: 7) (7) accacacacaaggcuacuucccugau;(SEQ ID NO: 8) (8) acagccgccuagcauuucaucac; (SEQ ID NO: 9) (9)ggauggugcuucaagcuaguaccaguu.

In the invention, conserved oligo-nucleotides sequences among all theHIV genome published were obtained by homology alignment. HIV geneexpression could be knocked down and HIV genome can be degraded when theRNA was introduced into mammalian cells. Pharmaceuticals derived fromthe conserved sequences can significantly decrease the drug resistantproblems resulted from genomic mutagenesis.

A set of RNA sequences, which may be modified by other nucleotide at the5′ or 3′ terminal. Usually UU were added at the 3′ end of the RNAfragment to assure the match between RNA with targeted RNA.

A set of hairpin RNA sequences for the control of HIV infection and forthe prevention and treatment of AIDS, the hairpin sequences were derivedby the hybridization of the sequences (SEQ ID No. 1˜SEQ ID No. 9) or therelevant segments at 5′ terminal with their complement sequences, inwhich RNA sequences and the complement sequences were linked by a noncomplement sequence. Hairpin-like RNA retains activity of RNAinterference, and is particular employed to express interfere RNA in thecell since it is a RNA molecular.

A set of DNA sequences or their fragments which is against HIV infectionand be used in the prevention and treatment of AIDS:

1) The DNA sequences or their fragments, which correspond to the RNAsequences shown above or their fragments (SEQ ID No. 1˜SEQ ID No. 9 intable 1); or correspond to the double strand RNA sequence formed byhybridization of RNAs shown above with its complement sequence, or,

2) The DNA sequences or their fragments, which correspond to the RNAsequences described in 1) or to their fragments which were modified attheir 5′ or 3′ by adding nucleotides; or

3) A single strand or double strand DNA sequence, which correspond tothe hairpin like RNA sequence as described above.

A set of expression vectors including both DNA vectors and RNA vectorsagainst HIV infection and used for the prevention or treatment of AIDS,in which RNA or DNA sequences described above were contained. InterfereRNA can be expressed when the vectors containing the DNA and RNAsequences mentioned above were introduced into cells under the controlof regulatory elements. The vectors include RNA vectors and DNA vectors.RNA vectors include but is not limited to retroviral vector, DNA vectorscarrying DNA sequences indicated and control elements include Plasmidand viral vectors such as adenovirus associated virus (AAV).

A set of liposomes against HIV infection and for the prevention andtreatment of AIDS, in which RNA, DNA sequences as well as the expressionvectors indicated above against HIV infection and for AIDS treatment andprevention were coated. Interfere RNA or vectors expressing interfereRNA was introduced into cell by the liposome indicated above.

The approach to fight against HIV infection and for AIDS prevention andtreatment, by which the above indicated RNAs, DNAs, expression vectorsor liposomes were introduced into eukaryotic cell lines, animals orhuman beings. E.g. Approaches employing liposome and viral vectors.

The application of the nucleotides in the prevention of HIV infectionand AIDS treatment. Pharmaceuticals for diagnosis, prevention andtreatment of HIV infection and AIDS were derived from the abovementioned RNAs, DNAs, Expression vectors, liposomes or approaches.

DESCRIPTIONS OF THE APPENDIX FIGURES

FIG. 1. Construction of report plasmid pEGFP-gp120.

FIG. 2 EGFP-gp120 expression was knocked down by double strand interfereRNA.

FIG. 3 EGFP-gp120 expression was knocked down by double strand interfereRNA as demonstrated by Western-Blot.

FIG. 4 The construction of p-H1-gp120i from which the hairpin RNA couldbe expressed in the cells.

FIG. 5 Construction of plasmid pAAV-120i.

FIG. 6 GFP-GP120 expression was knocked down by hairpin-like doublestrand RNA expressed by recombinant AAV.

BEST APPROACHES TO REALIZE THE INVENTION

All the protocols are generally based on the protocols described inMolecular Cloning, 3^(rd) edition.

Example 1 Most Conserved HIV RNA Sequence

HIV genome sequences published were selected and separated into 70 ntfragments based on functional genes of HIV. Homology of every fragmentwith more than 140,000 sequences in Genebank (National Center ofBiological Information, USA), EMBL (Nucleotide Sequence Database inEurope Molecular Biology Laboratory), DDBJ (Japan nucleotide database)and GDB (gene database) was analyzed by BlastN 2.2.4/2.2.5. Theconserved RNA sequences were selected by the following criteria: (1) Thesequence is equal or longer than 19 nt; (2) The sequence was 100%homology with at least 1000 HIV sequences in the database; (3) If 100%homology fragments can not be found, The sequences containing 1mismatched nucleotide were included. The results of the analysis wereshown in table 1 and table 2.

TABLE 1 Most conserved HIV RNA sequences found by homology analysis NoHIV gene RNA sequence 1 gag-pol Aucaaugaggaagcugcagaaugg (SEQ ID NO: 1)2 gag-pol Gggaagugacauagcaggaacuacuag (SEQ ID NO: 2) 3 gag-poluaaauaaaauaguaagaauguauagcccu (SEQ ID NO: 3) 4 env Uaugggguaccugugugga(SEQ ID NO: 4) 5 env Gccaauucccauacauuauugugc (SEQ ID NO: 5) 6 EnvUuaaauggcagucuagcagaa (SEQ ID NO: 6) 7 Nef Accacacacaaggcuacuucccugau(SEQ ID NO: 7) 8 3-UTR Acagccgccuagcauuucaucac (SEQ ID NO: 8) 9 LTRGgauggugcuucaagcuaguaccaguu (SEQ ID NO: 9)

TABLE 2 Homology analysis of the conserved RNA sequences with sequencesin database Sequence Fragment HIV 100% (s) with size sequence homology 1nt No HIV gene (nt) compared sequences mismatch 1 Gag-pol 24 1050 1050 02 Gag-pol 27 1051 1050 1 3 Gag-pol 29 1050 1048 2 4 env 19 1050 1050 0 5env 24 1050 1050 0 6 env 21 1050 1050 0 7 nef 26 1082 1082 0 8 3-UTR 231070 1070 0 9 LTR 27 1069 1069 0

Example 2 HIV env Gene Expression was Knocked Down by ChemicallySynthesized Double Strand RNA

Positive and negative (complement strand) RNA strand were synthesizedaccording to the SEQ ID#1 with UU modification at 3′ of the sequences.

(SEQ ID NO: 10) 5′ uaugggguaccuguguggauu (SEQ ID NO: 11) 3′uuauaccccauggacacaccu

As showed in FIG. 1, plasmid pEGFPCI (Clontech, Calif.) was doubledigested with EcoRI and BamHI at 37° C. for 1 hour. Large fragment wasextracted and was used as vector; HIV gp120 gene was obtained by PCRusing 2 ng HIV cDNA(Bru strain) as template plus gp120 primers(A:5′cggaattctaaagagcacaaga cagtggac, (SEQ ID NO:12) B: 5′cggatcctactctaccgtcagcgtcattga (SEQ ID NO:13) 100 ng each) in a buffercontaining 2.5u Pfu high fidelity DNA polymerase, dNTP 250 μmol/L, 2.5mmol/L MgCl₂, 25 mmol/L TrisHCl(pH8.3). Polymerase chain reaction (PCR)was carried out using Perkin Elmer 9700 thermocycler (94° C. 30s, 50° C.30s, 72° C. 90s, 30 cycles), DNA fragment resulted PCR was doubledigested by EcoRI and BamHI(Biolabs) after being purified by Qiagen GelExtraction Kit and ligated with the vector described above. The ligatedmixture was transformed into E. coli JM109 (Promega), and the plasmidpEGFP-gp120 was obtained. Fusion protein of GFP and HIV gp120 should beexpressed by transfection of the plasmid into mammalian cells.

HEK 293 cells (from ATCC) were co-transfected with 1 μg plasmidpEGFP-gp120 and 1 μg double strand RNA described above usingLIPOFECTamine (rf. Manual from Invitrogen), The cells were assayed byfluorescent microscopy and the cell lysate were analyzed byimmuno-blotting with anti-GFP antibody (Clontech) 36 h aftertransfection. A mock double strand RNA (rf. Ds RNA correspond HIV GAGgene, see EXAMPLE 3) was employed as control.

Results: As shown in FIG. 2, expression of the fusion protein wasknocked down by env specific double strand RNA compared to the control.The experiment was repeated twice, and was shown as DsRNA1 and DsRNA2respectively. As shown in FIG. 3, the expression level of GFP-HIV GP120fusion protein was knocked down up to 80%.

Example 3 HIV gag Gene Expression was Knocked Down by Synthesized DoubleStrand RNA

Based on the conserved gag RNA sequence (Seq ID#2 in table 1), a 21 ntoligonucleotides and its complement sequence was synthesized. Thesequences contain 19 nt from Seq ID#2 and two U at 3′ of each fragment.Double strand RNA was obtained by annealing.

(SEQ ID NO: 14) 5′ gugacauagcaggaacuacuu (SEQ ID NO: 15) 3′uucacuguaucguccuugaug

Gag gene from HIV (LAV-1, Bru isolate) was amplified and cloned intopEGFP C1 vector (Clontech, Calif.) as described in EXAMPLE 2, GFP-HIVgag fusion protein was expected to be expressed by the plasmid when itwas transfected into cells.

The plasmid as well as double strand RNA was co-transfected into HEK 293cells by LIPOFECTamine protocol, GFP-HIV gag protein was demonstrated tobe knocked down by the double strand RNA compared to the mock doublestrand, as shown by the fluorescent microscopy of the cells 36 h aftertransfection.

Example 4 Nef Gene Expression was Knocked Down by Synthesized DoubleStrand RNA

According to the conserved nef sequence (SEQ ID#7 in table 1), a 21 ntoligo-nucleotide was synthesized with it complement RNA sequence, inwhich the 5′ 19 nt was derived from SEQ ID#7 and two U was added to the3′ of each oligo-nucleotide. Double strand RNA was obtained byannealing.

(SEQ ID NO: 16) 5′ accacacacaaggcuacuuuu (SEQ ID NO: 17) 3′uuugguguguguuccgaugaa

Gene encoding nef protein was amplified and cloned into pEGFPC1 as shownin example 2, and the GFP-Nef fusion protein was expected to beexpressed by the cells containing the recombinant plasmid.

HEK 293 cells were co-transfected with the plasmid obtained and thedouble strand RNA synthesized, it has demonstrated that the expressionof the GFP-HIV nef fusion protein was knocked down by the nef specificdouble strand RNA as compared to the mock double strand RNA, as shown byfluorescent microscopy 36 hours after transfection.

Example 5 Expression of Other HIV Proteins Could be Knocked Down bySynthesized Double Strand RNA (Table 3)

Table 3 Expression of other HIV genes were knocked down by the novodouble strand RNA

Targeted Efficacy of No DsRNA HIV gene inhibition 1 5′aucaaugaggaagcugcaguu gag-pol ++++ (SEQ ID NO: 18) 3′uuuaguuacuccuucgacguc (SEQ ID NO: 19) 2 5′ guaagaauguauagcccuguu gag-pol+++ (SEQ ID NO: 20) 3′ uucauucuuacauaucgggac (SEQ ID NO: 21) 3 5′uucccauacauuauugugcuu env +++ (SEQ ID NO: 22) 3′ uuaaggguauguaauaacacg(SEQ ID NO: 23) 4 5′ aaauggcagucuagcagaauu env +++ (SEQ ID NO: 24) 3′uuuuuaccgucagaucgucuu (SEQ ID NO: 25)

 +++ 60-80% inhibition; ++++ 80-100% inhibition.

Example 6 Expression of HIV Envelope was Knocked Down by RNAi Expressedby Eukaryotic Vector Containing Double DNA Fragments Encoding ConservedHairpin SiRNA

DNA corresponding to the fragment of SeqID#5 RNA sequence shown at table1 and its hybrid sequence (bold italic) were synthesized, double strandDNA fragment was obtained by annealing. BamHI and HindIII sites wereincluded at its 5′ and 3′, respectively. There are 9 bp space betweenconserved sequence and its hybridization sequence. Fragment B is thecomplement sequence of fragment A:

(SEQ ID NO: 26) A: 5′ gatcccc

ttcaagaga

tttttggaaa (SEQ ID NO: 27) B: 5′ agcttttccaaaaa

tctcttgaa

ggg

As shown in FIG. 4, Human H1 promoter was amplified by primer 1(5′-TAATTAATGCGGCCGCAATTCGAACGCTGACGTC-3′) (SEQ ID NO:28) and primer 2(5′-GCACTAGTAAGCTTGGATCCGTGGTCTCATACAGAACTTATAAGATTCCC-3′ (SEQ ID NO:29)using 1 μg human genomic DNA as templates and cloned into Asel and Xbalsites of plasmid pEGFP (Clontech). The ligated mixture was transformedinto E. coli JM109, and the recombinant plasmid pH1 was obtained.Annealed double strand DNA fragment described was cloned into pH1 at itsBamHI and HindIII sites, and a new recombinant plasmid, pH1-gp120i, wasobtained. Hairpin RNA could be transcribed by RNA polymerase III in thecells harboring pH1-gp120i.

HEK293 cells were co-transfected with 4 μg pH1-gp120i plasmid (sameamount of pH1 was used as control) and a plasmid expressing EGFP-HIVGP120. The differential expression of EGFP-HIV GP120 was assayed asdescribed in Example 2. The results demonstrated that RNAi encoded byplasmid containing DNA fragment encoding hairpin RNA can effectivelyinhibit the expression of target HIV gene.

Example 7 Expression of HIV GP120 was Knocked Down by RNAi Transcribedin the Cells Infected by Adenovirus Associated Virus (AAV) which ContainH1 Promoter and the Relevant DNA Fragment Encoding Hairpin RNA asDescribed in Example 6

As shown in FIG. 5, plasmid pAAV-MCS (Stratagene) was digested with NotIand HindIII; DNA fragment containing H1 promoter and DNA fragmentencoding hairpin RNA corresponded to gp120 was obtained by digestingpH1-gp120 with NotI and HindIII. The fragment was ligated to vector byT4 DNA ligase, and plasmid pAAV-gp120i was constructed. HEK 293FT cellswere co-transfected with the plasmid (4 μg), helping plasmid pHelper (1μg, Stratagene) and plasmid pAAV-RC (2 μg Stratagene) by LIPOFECTamine,and empty vector (pAAV-MCS) was used as control. Recombinant AAV andcontrol AAV was harvested 48 hour after transfection.

HEK 293 cells were transfected by pEGFP-GP120 (1 μg) as described andinfected by the recombinant AAV encoding RNAi or empty AAV, fluorescentof GFP expressed was assayed 24 h after infection by fluorescentmicroscope.

As shown in FIG. 6, GFP-GP120 expression was significantly inhibited bythe recombinant AAV which encoded hairpin RNA.

Industrial Applicability

The invention was superior to the current technology as shown below:

Highly conserved RNA fragments in all published HIV genome were obtainedby homology analysis. Double strand RNA derived from the highlyconserved RNA could effectively knock down the expression of HIV gene.HIV gene expression could also inhibited by dsRNA encoded by plasmid aswell as recombinant adenovirus associated virus containing correspondedDNA sequence.

<110> Beijing Joinn Pharmaceutical Center

<120> A Set of Oligo-Nucleotides Against HIV Infection and ItsApplication in the Prevention and Treatment of Acquired ImmuneDeficiency Syndrome

<130>

<160> 9

<170> PatentIn version 3.1

<210> 1

<211> 24

<212> RNA

<213> Lentivirus genera

<400> 1

aucaaugagg aagcugcaga augg 24<210> 2<211> 27<212> RNA<213> Lentivirus genera<400> 2

gggaagugac auagcaggaa cuacuag 27<210> 3<211> 29<212> RNA<213> Lentivirus genera<400> 3

uaaauaaaau aguaagaaug uauagcccu 29<210> 4<211> 19<212> RNA<213> Lentivirus genera<400> 4

uaugggguac cugugugga 19<210> 5<211> 24<212> RNA<213> Lentivirus genera<400> 5

gccaauuccc auacauuauu gugc 24<210> 6<211> 21<212> RNA<213> Lentivirus genera<400> 6

uuaaauggca gucuagcaga a 21<210> 7<211> 26<212> RNA<213> Lentivirus genera<400> 7

accacacaca aggcuacuuc ccugau 26<210> 8<211> 23<212> RNA<213> Lentivirus genera<400> 8

acagccgccu agcauuucau cac 23<210> 9<211> 27<212> RNA<213> Lentivirus genera<400> 9

ggauggugcu ucaagcuagu accaguu 27

1. An isolated nucleic acid molecule selected from the group consistingof: a single stranded RNA consisting of SEQ ID NO:3; a single strandedRNA consisting of SEQ ID NO:3 with two uracil nucleotides appended tothe 5′ terminus, the 3′ terminus, or both; an siRNA comprising a doublestranded RNA consisting of SEQ ID NO:3 annealed to its complementaryribonucleotide sequence; and an siRNA comprising a double stranded RNAconsisting of SEQ ID NO: 3 annealed to its complementary ribonucleotidesequence, with two uracil nucleotides appended to each 3′-terminus, each5′-terminus, or both.
 2. The nucleic acid molecule of claim 1, whereinsaid RNA is a single stranded RNA consisting of SEQ ID NO:3 with twouracil nucleotides appended to the 5′-terminus, the 3′-terminus, orboth, or a double stranded RNA consisting of SEQ ID NO: 3 annealed toits complementary ribonucleotide sequence, with two uracil nucleotidesappended to each 3′-terminus, each 5′-terminus, or both.
 3. An isolatedhairpin RNA consisting of a stem part and a loop part, wherein said stempart is a double stranded RNA consisting of SEQ ID NO: 3 annealed to itscomplementary ribonucleotide sequence, or which stem part is a doublestranded RNA consisting of SEQ ID NO: 3 annealed to its complementaryribonucleotide sequence with two uracil nucleotides appended to the5′-terminus, the 3′-terminus, or both, and wherein said loop is anon-complementary spacer.
 4. An isolated single-stranded ordouble-stranded DNA, wherein: 1) said single-stranded DNA or one strandof said double-stranded DNA encodes the RNA or siRNA of claim 1, or itscomplementary sequence; or 2) said single-stranded DNA or one strand ofsaid double-stranded DNA encodes the RNA or siRNA of claim 2, or itscomplementary sequence; or 3) said single-stranded DNA or one strand ofsaid double-stranded DNA encodes the RNA of claim 3, or itscomplementary sequence.
 5. An expression vector encoding the RNA of anyone of claims 1-3.
 6. A liposome encapsulating the RNA of any one ofclaims 1-3.
 7. A liposome encapsulating the vector of claim
 5. 8. Anexpression vector comprising the DNA of claim
 4. 9. A liposomeencapsulating the DNA of claim
 4. 10. A liposome encapsulating thevector of claim 8.